A comparative investigation of the thermal unfolding of pseudoazurin in the Cu(II) holo and apo form

The contribution of the copper ion to the stability and to the unfolding pathway of pseudoazurin was investigated by a comparative analysis of the thermal unfolding of the Cu(II)-holo and apo form of the protein. The unfolding has been followed by calorimetry, fluorescence, optical density and electron paramagnetic resonance (EPR) spectroscopy. The thermal transition of Cu(II)-holo pseudoazurin is irreversible and occurs between 60.0 and 67.3 °C, depending on the scan rate and technique used. The denaturation pathway of Cu(II)-holo pseudoazurin can be described by the Lumry-Eyring model: N  U  F; the protein reversibly goes from the native (N) to the unfolded (U) state, and then irreversibly to the final (F) state. The simulation of the experimental calorimetric profiles, according to this model, allowed us to determine the thermodynamic and kinetic parameters of the two steps. The G value calculated for the Cu(II)-holo pseudoazurin is 39.2 kJmol-1 at 25 °C. The sequence of events in the denaturation process of Cu(II)-holo pseudoazurin emerging starts with the disruption of the copper site and the hydrophobic core destabilization followed by the global protein unfolding. According to the EPR findings, the native type-1 copper ion shows type-2 copper features after the denaturation. The removal of the copper ion (apo form) significantly reduces the stability of the protein as evidenced by a G value of 16.5 kJmol-1 at 25 °C. Moreover, the apo Paz unfolding occurs at 41.8 °C and is compatible with a two state reversible process N  U.